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CircMIIP Contributes to Non-Small Cell Lung Cancer Progression by Binding miR-766-5p to Upregulate FAM83A Expression

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Abstract

Background

Circular RNA migration and invasion inhibitory protein (circMIIP) is reported to be upregulated in non-small cell lung cancer (NSCLC) tissues compared with normal tissues. However, the role and working mechanism of circMIIP in NSCLC progression remain largely unclear.

Methods

Cell proliferation ability was analyzed by colony formation assay, cell counting kit-8 (CCK-8) assay, and 5-ethynyl-2′-deoxyuridine assay. Cell apoptosis was assessed by flow cytometry. Transwell assays were performed to analyze the migration and invasion abilities of NSCLC cells. The interaction between microRNA-766-5p (miR-766-5p) and circMIIP or family with sequence similarity 83A (FAM83A) was validated by dual-luciferase reporter assay and RNA immunoprecipitation assay. Xenograft tumor model was established to analyze the role of circMIIP on tumor growth in vivo.

Results

CircMIIP was highly expressed in NSCLC tissues and cell lines. CircMIIP knockdown restrained the proliferation, migration and invasion and induced the apoptosis of NSCLC cells. CircMIIP acted as a molecular sponge for miR-766-5p, and circMIIP silencing-mediated anti-tumor effects were largely overturned by the knockdown of miR-766-5p in NSCLC cells. miR-766-5p interacted with the 3’ untranslated region (3′UTR) of FAM83A, and FAM83A overexpression largely reversed miR-766-5p accumulation-induced anti-tumor effects in NSCLC cells. CircMIIP competitively bound to miR-766-5p to elevate the expression of FAM83A in NSCLC cells. CircMIIP knockdown significantly restrained xenograft tumor growth in vivo.

Conclusion

CircMIIP promoted cell proliferation, migration and invasion and suppressed cell apoptosis in NSCLC cells through mediating miR-766-5p/FAM83A axis.

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Data Availability

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

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Acknowledgements

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Authors and Affiliations

  1. Department of Thoracic Surgery, Affiliated Hospital of Guizhou Medical University, No. 28 Guiyi Street, Yungang District, Guiyang, 550004, Guizhou, China

    Tao Wang, Xu Zhu & Kai Wang

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  1. Tao Wang
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Contributions

All authors made substantial contribution to conception and design, acquisition of the data, or analysis and interpretation of the data; take part in drafting the article or revising it critically for important intellectual content; gave final approval of the revision to be published; and agree to be accountable for all aspect of the work. Conceptualization, Methodology, Formal analysis and Data curation: XZ and KW; Validation and Investigation: TW and XZ; Writing—original draft preparation and Writing—review and editing: TW, XZ and KW; Approval of final manuscript: all authors.

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Correspondence to Tao Wang.

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The present study was approved by the ethical review committee of Affiliated Hospital of Guizhou Medical University. Written informed consent was obtained from all enrolled patients.

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Supplementary Information

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Supplementary file1 (DOCX 14 kb)

408_2021_500_MOESM2_ESM.tif

Supplementary file2 Sup. Fig 1. CircMIIP is highly expressed in NSCLC tissues and cells. (A) Schematic illustration showing the genomic location of circMIIP generated from its host gene MIIP. (B, C) CircMIIP expression was measured in NSCLC tissues and normal tissues, as well as in NSCLC cell lines (NCI-H23 and A549) and human lung epithelial cell line (BEAS-2B) using RT-qPCR. *P < 0.05 (TIF 3420 kb)

408_2021_500_MOESM3_ESM.tif

Supplementary file3 Sup. Fig 2. CircMIIP silencing restrains the proliferation of NSCLC cells. (A and B) NSCLC cells were transfected with si-NC or si-circMIIP#3. Cell proliferation ability was analyzed by colony formation assay and CCK-8 assay. *P < 0.05 (TIF 3017 kb)

408_2021_500_MOESM4_ESM.tif

Supplementary file4 Sup. Fig 3. The representative images of EdU assay, flow cytometry, and transwell assays in Figure 1. (A) The representative images of EdU assay in Figure 1B. (B) The representative images of flow cytometry in Figure 1C. (C and D) The representative images of transwell assays in Figure 1D and 1E (TIF 22548 kb)

408_2021_500_MOESM5_ESM.tif

Supplementary file5 Sup. Fig 4. CircMIIP absence restrains the proliferation of NSCLC cells largely by up-regulating miR-766-5p. (A and B) NSCLC cells were transfected with si-circMIIP#3 alone or together with anti-miR-766-5p. Colony formation assay and CCK-8 assay were conducted to analyze the proliferation ability of NSCLC cells. *P < 0.05 (TIF 761 kb)

408_2021_500_MOESM6_ESM.tif

Supplementary file6 Sup. Fig 5. miR-766-5p overexpression suppresses the proliferation of NSCLC cells largely by down-regulating FAM83A. (A and B) NSCLC cells were transfected with miR-766-5p alone or together with FAM83A. Cell proliferation ability was analyzed by colony formation assay and CCK-8 assay. *P < 0.05 (TIF 753 kb)

408_2021_500_MOESM7_ESM.tif

Supplementary file7 Sup. Fig 6. CircMIIP/miR-766-5p/FAM83A axis in established in BEAS-2B cells. (A) The knockdown efficiencies of three circMIIP-targeted siRNAs were analyzed in BEAS-2B cells by RT-qPCR. (B) BEAS-2B cells were transfected with si-NC or si-circMIIP#3, and CCK-8 assay was conducted to analyze the proliferation ability of transfected BEAS-2B cells. (C and D) Dual-luciferase reporter assay and RIP assay were conducted to verify the target relationship between circMIIP and miR-766-5p in BEAS-2B cells. (E and F) The interaction between miR-766-5p and FAM83A in BEAS-2B cells was confirmed by dual-luciferase reporter assay and RIP assay. *P < 0.05 (TIF 1432 kb)

408_2021_500_MOESM8_ESM.tif

Supplementary file8 Sup. Fig 7. miR-766-5p overexpression has almost no effect on the proliferation of BEAS-2B cells. (A and B) BEAS-2B cells were transfected with miR-NC or miR-766-5p. (A) RT-qPCR was conducted to analyze the overexpression efficiency of miR-766-5p mimics in BEAS-2B cells. (B) CCK-8 assay was performed to assess the proliferation of BEAS-2B cells. *P < 0.05 (TIF 460 kb)

408_2021_500_MOESM9_ESM.tif

Supplementary file9 Sup. Fig 8. A schematic diagram shows the role of circMIIP/miR-766-5p/FAM83A axis on the malignant behaviors of NSCLC cells (TIF 3065 kb)

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Wang, T., Zhu, X. & Wang, K. CircMIIP Contributes to Non-Small Cell Lung Cancer Progression by Binding miR-766-5p to Upregulate FAM83A Expression. Lung 200, 107–117 (2022). https://doi.org/10.1007/s00408-021-00500-3

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